It is proposed to investigate the mechanism of membrane fusion at the molecular level. Our research plan is an inductive approach which utilizes relatively simple but well-defined model systems where the participation of individual membrane components can be studied in detail. The membranes to be studied include phospholipid vesicles of varying composition and size with and without incorporated proteins, several secretory vesicles, erythrocyte ghosts and erythrocyte-derived, right-side out and inside-out vesicles. We propose to examine the role of calcium and other divalent metals; the importance of specific phospholipid composition and lateral lipid phase separations; the ability of various purified proteins and other putative fusogens (such as lysolecithin, calcium phosphate, polyethylene glycol, etc.) to either initiate or enhance fusion; the similarity and differences in fusion characteristics between various natural membrane vesicles and lipid vesicles of defined composition. Fusion will be followed by a newly established sensitivie fluorescence assay, which permits a detailed study of the kinetics of mixing of vesicle aqueous contents. These observations will be correlated with simultaneous measurements of rates of aggregation and release of vesicle contents, and with parallel studies of metal ion and protein binding, E.M. morphological observations and other physical techniques involving microcalorimetry and X-ray diffraction. Our relatively simple systems will methodically be expanded in terms of their complexity to approximate more closely membrane fusion phenomena at the cellular level.